ORIGINAL RESEARCH ARTICLE published: 25 November 2014 doi: 10.3389/fmicb.2014.00591

Multiple sulfur isotope signatures of sulfite and thiosulfate reduction by the model dissimilatory sulfate-reducer, Desulfovibrio alaskensis str. G20 William D. Leavitt 1,2*, Renata Cummins 1 , Marian L. Schmidt 1,3 , Min S. Sim 4,5 , Shuhei Ono 4 , Alexander S. Bradley 2 and David T. Johnston 1* 1 2 3 4 5

Department of Earth and Planetary Sciences, Harvard University, Cambridge, MA, USA Department of Earth and Planetary Sciences, Washington University in St. Louis, St. Louis, MO, USA Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI, USA Department of Earth, Atmosphere and Planetary Science, Massachusetts Institute of Technology, Cambridge, MA, USA Division of Geological Sciences, California Institute of Technology, Pasadena, CA, USA

Edited by: Partha Basu, Duquesne University, USA Reviewed by: John W. Moreau, University of Melbourne, Australia Mustafa Yucel, GEOMAR Helmholtz Centre for Ocean Research Kiel, Germany *Correspondence: William D. Leavitt, Department of Earth and Planetary Sciences, Washington University in St. Louis, Campus Box 1169, Rudolph Hall, 1 Brookings Drive, St. Louis, 63130 MO, USA e-mail: [email protected]; David T. Johnston, Department of Earth and Planetary Sciences, Harvard University, Hoffman Labs, 302, 20 Oxford Street, Cambridge, 02138 MA, USA e-mail: [email protected]

Dissimilatory sulfate reduction serves as a key metabolic carbon remineralization process in anoxic marine environments. Sulfate reducing microorganisms can impart a wide range in mass-dependent sulfur isotopic fractionation. As such, the presence and relative activity of these organisms is identifiable from geological materials. By extension, sulfur isotope records are used to infer the redox balance of marine sedimentary environments, and the oxidation state of Earth’s oceans and atmosphere. However, recent work suggests that our understanding of microbial sulfate reduction (MSRs) may be missing complexity associated with the presence and role of key chemical intermediates in the reductive process. This study provides a test of proposed metabolic models of sulfate reduction by growing an axenic culture of the well-studied MSRs, Desulfovibrio alaskensis strain G20, under electron donor limited conditions on the terminal electron acceptors sulfate, sulfite or thiosulfate, and tracking the multiple S isotopic consequences of each condition set. The dissimilatory reduction of thiosulfate and sulfite produce unique minor isotope effects, as compared to the reduction of sulfate. Further, these experiments reveal a complex biochemistry associated with sulfite reduction. That is, under high sulfite concentrations, sulfur is shuttled to an intermediate pool of thiosulfate. Site-specific isotope fractionation (within thiosulfate) is very large (34 ε ∼ 30) while terminal product sulfide carries only a small fractionation from the initial sulfite (34 ε < 10): a signature similar in magnitude to sulfate and thiosulfate reduction. Together these findings show that microbial sulfate reduction (MSR) is highly sensitive to the concentration of environmentally important sulfur-cycle intermediates (sulfite and thiosulfate), especially when thiosulfate and the large site-specific isotope effects are involved. Keywords: microbial sulfate reduction, multiple sulfur isotopes, biogeochemical sulfur cycle, thionates, sulfur intermediates

INTRODUCTION The geological record preserves only select snapshots of paleoenvironments. One of the more robust continuous records of paleo-redox is stored in sedimentary sulfide and sulfate minerals (Thode et al., 1961; Holland, 1973; Strauss, 1997, 1999; Canfield and Raiswell, 1999; Canfield, 2004; Alroy et al., 2008). The isotopic composition of these phases may serve as a prominent proxy for the oxidation state of Earth surface environments (Berner and Canfield, 1989; Kurtz et al., 2003; Hayes and Waldbauer, 2006; Halevy et al., 2012). For instance, the partial pressure of oxygen in Earth’s atmosphere is thought to control concentrations of dissolved sulfate and oxygen in the oceans, which in turn may be recorded by the difference in the isotopic compositions between sulfate and sulfide minerals (Canfield, 2001a,b; Habicht and Canfield, 2001; Habicht et al., 2002). At the core

www.frontiersin.org

of these interpretations is an understanding of the isotope fractionations associated with the numerous redox reactions that characterize the modern sulfur cycle. Among the biologically mediated of sulfur redox reactions is MSR, coupling the oxidation of organic matter or hydrogen to the reduction of sulfate (Peck, 1959, 1962; Rabus et al., 2006; Bradley et al., 2011). MSR is responsible for a large proportion of the organic matter remineralization in anoxic environments (Jorgensen, 1982; Bowles et al., 2014), making it a key environmental process and an important link between the cycles of sulfur carbon and oxygen. MSR is also capable of producing a wide range of mass-dependent sulfur isotope fractionations (Canfield et al., 2010; Johnston, 2011; Sim et al., 2011a; Leavitt et al., 2013). In order to interpret the sulfur isotope variability within geological records—and as it relates to environmental conditions—we first need to understand the

November 2014 | Volume 5 | Article 591 | 1

Leavitt et al.

controls on the fractionation of sulfur isotopes within the MSR pathway. Studies of the sulfate reduction metabolism often converge on the idea that sulfate and electron donor availability control the rates of reduction, and in turn, the expressed isotopic fractionation (Harrison and Thode, 1958; Kaplan and Rittenberg, 1964; Chambers et al., 1975; Goldhaber and Kaplan, 1975; Canfield, 2001b; Habicht et al., 2002, 2005; Bradley et al., 2011; Sim et al., 2012; Leavitt et al., 2013). Simply, these variables determine the capacity to deliver reductant to the respiratory reaction network and the relative rates at which electrons and S-bearing oxidants are supplied to catabolic enzymes. The isotopic fractionation associated with MSR is visualized through a schematic depiction (Figure 1A) of the central metabolism (Rees, 1973; Brunner and Bernasconi, 2005; Bradley et al., 2011). This schematic has evolved as our understanding of the metabolism has become more biochemically informed, beginning with a simple three step process (Harrison and Thode, 1958), through to a revised

FIGURE 1 | Different views of the sulfate reduction metabolic network. (A) The model of dissimilatory sulfate reduction modified from Bradley et al. (2011). As it relates to this study, the schematic outlines the potential for numerous reactions between sulfite/bisulfite 2− − (HSO− 3 /SO3 ) and hydrogen sulfides/bisulfide (H2 S/HS ) (insets B–D). As the isotopic consequences of most of these potential reactions are unknown or under-constrained, this work aims to better assay both the isotope effects and conditions that favor the production of some of these intermediates. Outlined here are the potential reactions for (B,C) sulfite and (D) thiosulfate [(S-SO3 )2− ] reduction, given the standard model of

Frontiers in Microbiology | Microbiological Chemistry

The isotopic consequences of sulfite and thiosulfate reduction

reaction series with the first thorough mathematic derivation (Rees, 1973), to one taking on a more involved reaction chain (Brunner and Bernasconi, 2005), and finally to the most recent update that incorporates a variety of biochemical and enzyme structural information (see Figure 1; Bradley et al., 2011), only available in the last few years (Oliveira et al., 2008, 2011; Venceslau et al., 2010). Much is gained through a close reading of the MSR network as presented in Figure 1. For example, the fractionations classically prescribed to this pathway are associated with sulfate uptake by the cell, the reduction of activated sulfate (APS: adenosine 5 -phosphosulfate) to sulfite, and the terminal reduction of sulfite to sulfide. Given older empirical limits from lab experiments, the maximum fractionation capacity was inferred as the sum of these three steps (with fractionation factors of +3, −25 and −25, respectively and 47 in total) (Harrison and Thode, 1958; Kaplan and Rittenberg, 1964; Rees, 1973; Chambers et al., 1975). However, recent work with pure and enrichment cultures demonstrated an increased fractionation capacity (up to

the biochemistry associated with sulfate (SO2− 4 ) reduction (A). Trithionate [(3 OS-S-SO3 )2− ] is included here, however not observed in this study. This is not an exhaustive depiction of the MSR network, though is a testable topological prediction that would be better informed with future biochemical inquiry (c.f. Venceslau et al., 2014). The valence of the S as it moves through the pathway: sulfate and APS (6+), sulfite (4+), outer S’s in trithionate (4+), central S in trithionate (2+), sulfonate S in thiosulfate (5+), reduced S in thiosulfate (0 to 1−), sulfide/hydrogen sulfide (2−). The values for trithionate are best estimates, while those for thiosulfate are from the literature (Vairavamurthy et al., 1993).

November 2014 | Volume 5 | Article 591 | 2

Leavitt et al.

66) of MSR (Canfield et al., 2010; Sim et al., 2011a; Leavitt et al., 2013), approaching theoretical predictions (∼74) from low temperature equilibrium exchange (Farquhar et al., 2003; Johnston et al., 2007). These observations require a careful reevaluation of isotopic fractionation and path sulfur follows during MSR. Determining fractionation factors within the MSR metabolism necessitates a multi-faceted approach. Classic isotope theory states that the kinetic isotope effects of a particular step are only expressed if that step is rate limiting within the reaction scheme (Hayes, 2001). Each reaction that outpaces the ratelimiting step will quantitatively transform its particular substrate to its product, precluding any isotopic discrimination. In reality, reaction chains (particularly in biological systems) do not behave so simply, as multiple steps may compete for rate limitation. The expressed fractionation associated with a particular reaction may instead be a function of how material is transported through a system (e.g., a linear vs. branched/vectorial pathway, Johnston et al., 2007; Bradley et al., 2011). Given these considerations, the network topology in Figure 1 provides a roadmap for identifying the significant reactions within MSR, highlighting where experimental work is most needed. As in Figure 1, sulfite availability serves as a major factor in determining both the reversibility of the MSR network and whether intermediates (such as thiosulfate) factor into the reaction scheme. Fortunately, the physiology of most cultured sulfate reducers carries some plasticity, allowing them to utilize MSR intermediates as terminal electron acceptors in place of sulfate. For instance, Desulfovibrio alaskensis strain G20 2− (G20) will reduce sulfite (SO2− 3 ) or thiosulfate (S2 O3 ) in lieu 2− of or in addition to sulfate (SO4 ) (Price et al., 2014). Here we present and discuss the results from closed-system (batch) experiments with an axenic culture of G20. For each experiment we account for sulfur isotope mass balance and project the major and minor S-isotope fractionation factors during the dissimilatory reduction of three different electron acceptors: sulfate, thiosulfate, and sulfite. Further, we compare these empirically derived values to calculations of the equilibrium fractionations between the relevant sulfur species. These data shed new light on the inner workings of the sulfate reduction metabolism, illustrate the range of isotopic potential intrinsic to these reactions, and capture a complex chemistry that blurs the lines between the classic picture of MSR and sulfur disproportionation reactions. The experiments in this study were designed to test our current understanding of how MSR behaves under conditions where the reaction intermediates sulfite or thiosulfate are available at high concentrations. This study tested the biochemistry, physiology, and isotopic fractionations associated with the MSR reaction network (Figure 1). In the classic models for MSR the sulfate anion is imported into the cell, where it can be either exported from the cell in a “back reaction” or activated to APS at the expense of cellular energy reserves (ATP; Peck, 1959). The fate of APS is similarly bidirectional, with both intracellular sulfate and sulfite as possible products (Peck, 1962), where the reduction of APS requires a two-electron gain and the oxidation a two-electron loss. Once as sulfite, there exist numerous possible reaction pathways (Bradley et al., 2011), some of which are directly tested in this study (see Results and Discussion).

www.frontiersin.org

The isotopic consequences of sulfite and thiosulfate reduction

The aim of this study was to determine the multiple sulfur isotope fractionation factors (33 α, 34 α) between reactant and product, during dissimilatory thiosulfate or sulfite reduction, as compared to control dissimilatory sulfate reduction experiments. This study was conducted with axenic cultures of D. alaskensis strain G20, and illustrates the metabolic potential of the MSR network laid out in Figure 1. We confirm growth with sulfate, sulfite and thiosulfate as the sole provided electron acceptors. In keeping with the thermodynamic treatment and recent observations in similar metabolic systems (Shirodkar et al., 2011), the presence of certain sulfoxy anions (e.g., thiosulfate) does not necessitate their reduction if a more energetically favorable electron acceptor is available (e.g., sulfite). Data and Discussion in support of this follow.

METHODS Experiments were conducted to determine the isotopic fractionation between electron acceptor (sulfate, sulfite or thiosulfate) and product sulfide during closed-system growth of D. alaskensis on lactate (electron donor). In addition, sulfate reduction experiments were conducted with either lactate or formate as the sole provided electron donor, whereas sulfite and thiosulfate reduction experiments were paired with lactate only. A pure culture of the sulfate-reducing bacterium D. alaskensis strain G20 was grown in airtight glass Balch tubes sealed with butyl rubber septa under a gas headspace of 90% nitrogen and 10% carbon dioxide. Media was degassed with this mix prior to inoculation. The medium consisted of (per liter) NaCl, 20 g; MgCl2 ·6H2 O, 3 g; CaCl2 ·2H2 O, 0.15 g; NH4 Cl, 0.25 g; KH2 PO4 , 0.2 g; KCl, 0.5 g; Na2 SeO4 ·10H2 O, 370 mg, as well as a vitamins and amino acids solution and a trace metals solution (Widdel and Bak, 1992). Electron acceptors were provided at 20 mM (sulfite or sulfate) or 10 mM (thiosulfate), initial concentrations), while electron donor was always provided at 10 mM (lactate or formate). All medium was prepared after solutions were degassed with O2 -free N2 for at least 1 h/L, and transferred in an anaerobic chamber under an atmosphere of N2 :H2 95:5. In each experiment un-inoculated controls were monitored for contamination, and a killed control to quantify any inoculum sulfur. All cultures were grown in their respective media for >10 transfers prior to the inoculation of the experiments reported here. Bacteria were transferred at a 1:100 dilution to guarantee the quantity of reduced sulfur carried over with the inoculum was below 10 mM. Each tube was sampled at roughly five time points spread throughout each experiment to ensure capture of exponential phase growth. At each sampling time (t), including at the start of the experiment (t = 0), optical density and chemistry measurements were performed. The optical density of each tube was measured at A600. These measurements were calibrated to absolute cell counts through a standard staining protocol (Moore et al., 1998). In parallel, concentrations of relevant chemical species were measured throughout. Sulfide was quantified colorimetrically (Cline, 1969) (detection limit of 50 μM, given the protocol used herein) whereas sulfate, thiosulfate and sulfite were all measured via ion chromatography (Leavitt, 2014) (detection limit 10 μM). Given the instability of sulfite under ambient atmosphere (O2 ), care was taken to avoid oxidation by preserving samples with formalin

November 2014 | Volume 5 | Article 591 | 3

Leavitt et al.

The isotopic consequences of sulfite and thiosulfate reduction

(0.1 mL of 600 mM anoxic formaldehyde added to each 1 mL of sample) (Leavitt, 2014). Sulfate concentrations were measured using an isocratic method, while sulfite and thiosulfate were measured using gradient elution. Trithionate was independently measured by cyanolysis (Kelly and Wood, 1994), though was never detectable (detection limit 40 μM). More sensitive methods for detecting trithionate or thiosulfate do exist, though were not employed in this study (Newton and Fahey, 1995). Elemental sulfur was detected as chromium reducible sulfide (CRS) (Canfield and Desmarais, 1994). Sulfur species were separated and prepared for major and minor isotope determination methods by established protocols (Leavitt et al., 2013; Leavitt, 2014). Sulfur isotope measurements of sulfate, sulfide and thiosulfate sulfur were performed first on a Thermo-Finnegan Delta V mass spectrometer, configured in continuous flow mode and connected to an Elemental Analyzer (measuring SO2 ). Given the above chemical methods, sulfate and sulfonate were measured as BaSO4 whereas sulfide and reduced thiosulfate S were measured as Ag2 S (data are reported in Tables 1–3), always with an excess of V2 O5 . From this, select data were chosen for high precision analyses via dual inlet on a Thermo Finnigan MAT 253 (as SF+ 5 ). Samples already as silver sulfide were fluorinated directly with an excess of pure F2 , cleaned cryogenically and via gas chromatography before introduction to the mass spectrometer. Samples as BaSO4 precipitates were chemically reduced to Ag2 S (Forrest and Newman, 1977) prior to high precision analyses. All isotope data presented herein is in standard delta notation, where the composition of a given sample is normalized (in our case) back to the original composition of the sulfoxy anion in the experiment. In the case of thiosulfate experiments, samples are normalized to the composition of the bulk S2 O2− 3 . This results in two distinct delta values—δ 33 S and δ 34 S (the ratios of 34 S/32 S in a standard relative to that of a reference). Values for δ 34 S are from both SO2 and SF6 measurements, whereas δ 33 S data are exclusively from SF6 measurements. When the composition of two reservoirs is being related, we define 34 ε [(34 α − 1) × 1000] and where 34 α between reservoirs A and B is (δ 34 SA /1000 + 1)/(d34 SB /1000 + 1). The triple isotope composition of a given reservoir can be related through: 33 S = δ 33 S − 1000 ×

   0.515  1 + δ 34 S/1000 −1 .

When two pools are being related in triple isotope space, we can use the slope of the line on a δ 33 S vs. δ 34 S plot, or: 33

λ = ln

33    α /ln 34 α .

For further information about the meaning and specific controls on mass-dependent fractionations, readers are referred to published discussions (Miller, 2002; Young et al., 2002; Farquhar and Wing, 2003; Johnston et al., 2005; Johnston, 2011). To calculate the fractionation factor of a given reaction, we apply a standard closed-system Rayleigh model with respect to the reactant:     (3x ) δ 34 SR, t = 0 + 1000 × ft α−1 − 1000. (1) δ 34 SR,t =

Frontiers in Microbiology | Microbiological Chemistry

where δ 34 SR, t is the isotopic composition (x = 3 or 4) of the reactant (R) though time (0, t). For this expression, the reaction coordinate is tracked with f, which represents the mole fraction of reactant remaining at time t. Additionally, the composition of a given product is calculated: δ 34 SP,t =

   [R]0 × δ 34 SR,t = 0 − ft × δ 34 SR,t   + [P]0 × δ 34 SP,t = 0 /[R]t . 

(2)

where the sulfur isotope composition of the reactant (R) or product (P) are related to their concentrations ([R] and [P]) at a given time point (t) (also see Equation 9 in Ono et al., 2012). Isotopic equilibrium can be calculated as the reduced partition function ratios of the different isotopologues (Bigeleisen and Mayer, 1947; Urey, 1947). For partition function calculations, 2− the necessary vibrational frequencies for H2 S, SO2− 3 , S2 O3 , and 2− SO4 were obtained by quantum chemical calculations. Herein we use the Gaussian03 software package to optimize geometry and calculate vibrational frequencies. The Hartree–Fock (HF) method and 6-31G∗ basis set without symmetry constraints were used for both geometry optimization and frequency calculation. As the HF theory is known to systematically overestimate fundamental frequencies (Scott and Radom, 1996), the scaling factor of 0.8928 was determined using a least-squares approach (Scott and Radom, 1996) and the experimental frequencies for H2 S (81), and applied to the calculated frequencies. To determine the shifts in vibrational frequencies upon isotopic substitution, the same calculation was made for each isotopologue in which one 32 S atom was replaced by 34 S. For all calculation processes, solution effects were included using the polarizable continuum model (PCM) (Miertus et al., 1981; Miertus and Tomasi, 1982).

RESULTS AND DISCUSSION Four discrete experiments were performed in this study. In each case, the dominant electron acceptor in the system was consumed with time as one or more reduced S-products accumulated. In two experiments G20 was grown on sulfate as the sole terminal electron acceptor and either lactate or formate as the sole electron donor. In the two additional experiments, only lactate was provided as the electron donor with either sulfite or thiosulfate as the sole provided terminal electron acceptor. For each, we quantify the loss of reactants and generation of products to explicitly track elemental and isotopic mass balance at each time point. Given the complex chemistry involved—for sulfite reduction in particular—we emphasize the requirement to explicitly measure each S pool at each time-point to close isotope massbalance. When coupled with optical densities and cell counts, cell specific reduction rates are also calculated (Detmers et al., 2001). The nature of the experiments (closed-system) requires additional calculations to determine the fractionation factor (3x α). Fortunately, our experimental methods circumvented an inoculum sulfide blank, streamlining previous mathematical treatments (Johnston et al., 2007). In the simplest case, the loss of a reactant and generation of a single product

November 2014 | Volume 5 | Article 591 | 4

Leavitt et al.

The isotopic consequences of sulfite and thiosulfate reduction

Table 1–4 | All geochemical, microbial, and isotopic data from batch experiments. Table 1

Sulfate

Sulfate

n (replicates) Lactate

Formate

Table 2

Thiosulfate

Table 3

Cells (106 /mL)

RSD (%)

f (%)

[SO4 ] (mM)

σ [SO4] (mM)

[HS− ] (mM)

σ [HS−] (mM)

34 ε

()

3

0

3.4

5.8

0.0

0.0

20.24

2.33

0.01

0.00

0.00

24

12.2

25.6

12.8

2.2

20.19

1.03

0.45

0.15

−4.02

3

29.4

71.6

35.2

142.3

6.9

19.24

0.21

1.39

0.43

−4.25

3

34.1

309.1

10.1

80.7

16.2

17.36

0.15

3.28

0.29

−3.87

3

39.5

423.9

1.3

61.8

18.9

16.56

0.15

3.82

0.12

−4.86

1

n.c.

6.0

20.06

0.00

1

k.c.

6.2

24.51

0.01

3

0

3.07

5.89

0.00

0.00

18.00

0.00

3

69

6.52

12.76

494.48

10.28

15.55

0.94

1.85

0.30

−6.55

3

81

17.69

34.16

420.70

21.80

14.38

1.33

3.92

0.54

−5.34

3

104.5

32.21

5.47

134.19

28.08

13.42

0.08

5.05

0.15

−5.88

1

n.c.

64.18

19.06

1

k.c.

6.20

18.20

Time (hours)

Cells (106 /mL)

RSD (%)

csSRR (f mol/cell*day)

f (%)

[S2 O3 ] (mM)

0.00

0.01 0.02

σ[S2O3] (mM)

[HS− ] (mM)

σ[HS−] (mM)

34

δ S[S*O3] δ 34 S[*S] () ()

δ 34 S[HS−] ()

3

0

1.54

0.56

0.00

0.00

18.00

0.07

0.00

0.00

2.16

−2.16

0.00

3

13

21.46

0.47

134.91

2.02

17.96

0.29

0.73

0.07

0.03

−2.81

−8.61

3

19

71.45

0.27

264.91

6.62

16.87

0.28

2.38

0.15

0.64

−2.61

−8.32

3

33

138.99

0.20

67.25

10.30

16.11

0.04

3.71

0.12

3.42

−1.26

−6.67

3

40

300.46

0.17

66.09

14.62

13.80

0.00

5.26

0.21

4.34

−1.46

−6.02

1

n.c.

0.06

0.00

1

k.c.

3.20

0.02

17.53 17.76

n Time Cells RSD csSRR fH2S (replicates) (hours) (106 /mL) (%) (f mol/cell*day) (%)

Sulfite Lactate

csSRR (f mol/cell*day)

3

n (replicates) Lactate

Time (hours)

fS2O3 (%)

[SO3 ] (mM)

σ[SO4] (mM)

[HS− ] (mM)

σ[HS−] [S2 O3 ] σ[S2O3] δ34 S[SO3] δ34 S[HS−] δ34 S[S*O3] δ34 S[*S] (mM) (mM) (mM) () () () ()

3

0

2.53

26.36

0.00

0.00

0.00

17.11

0.50

0.06

0.02

0.04

0.00

0.00

0.00

0.00

3

20

36.55

9.59

141.65

1.99

8.56

15.10

0.15

0.34

0.02

0.73

0.09

0.13

−10.27

0.00

0.00 0.00

3

23.4

108.29

5.14

452.96

4.91

21.47

12.80

0.08

0.84

0.07

1.84

0.14

0.41

−6.14

16.78

−15.19

3

26.5

227.33

3.58

408.33

9.70

37.20

9.66

0.16

1.66

0.06

3.18

0.12

1.29

−3.16

15.38

−13.62

3

30.6

469.77

0.30

180.89

15.55

52.12

5.92

0.60

2.66

0.20

4.46

0.82

0.61

−2.54

14.16

−13.29

1

n.c.

1.56

1

k.c.

Table 4

0.20 Sulfate

Sulfite

18.23

0.02

0.04

16.72

0.02

0.04

Sulfide

Sulfonate

Reduced S

Elemental sulfur

δ34 S

33 S

δ34 S

33 S

δ34 S

33 S

δ34 S

33 S

δ34 S

33 S

δ34 S

33 S

Sulfate

Lactate

0.27

−0.02





−3.95

0.008













Sulfate

Formate

1.76

−0.01





−5.15

0.016













Sulfite

Lactate

Thiosulfate

Lactate





1.29

0.033

−3.16

0.012

15.38

0.023

−13.62

0.002









0.61

0.038

−2.54

0.022

14.16

0.041

−13.29

0.041

















2.16

−0.037

−2.16

0.039













−6.67

0.013

3.42

−0.017

−1.26

0.006

−5.58

0.019









−6.02

0.024

4.34

−0.005

−1.46

0.009





For the two sulfate (Table 1) experiments, the corrected fractionation factors are presented

( 34 ε).

For thiosulfate and sulfite experiments (Tables 2, 3), where unique

fractionations between product and reactant can be calculated, values are noted as ( δ34 S are reported, 34 ε are calculated in the text), whereas when values are more difficult to ascertain, the direct isotopic compositions of the measured pools are reported. See text for full Discussion and equations for each. Note that reported thiosulfate concentrations are for total sulfur. Minor isotope data is presented in Table 4 for the time point(s) characterized, all relative to VCDT.

pool is then determined through a standard closed-system “Rayleigh” fractionation model (Nakai and Jensen, 1964) (Equations 1 and 2). It is through the use of these two expressions that we define isotope fractionation effects in our experiments.

www.frontiersin.org

SULFATE REDUCTION Sulfate reduction experiments with both lactate and formate generated sulfide as the lone S-bearing product. Near the end of exponential phase growth, 28.1 and 18.9% of the available sulfate had been consumed by formate and lactate oxidation,

November 2014 | Volume 5 | Article 591 | 5

Leavitt et al.

respectively (Figure 2A). This results in cell specific sulfate reduction rates (csSRRs) during exponential phase ranging up to 142 and 494 f mol per cell per day for formate and lactate oxidation, respectively. These rates are calculated from the change in sulfate concentrations between adjacent time points, divided by the difference in average cell count during that interval (Detmers et al., 2001). The same calculation can be performed with the product sulfide concentrations. These results are consistent with csSRR for related strains grown in batch (cf. Harrison and Thode, 1958). The fractionation associated with sulfate reduction was calculated using Equation (1), described above. For the formate oxidation experiment, the net 34 εSO4/H2S averaged −4.25 ± 0.44, whereas growth on lactate resulted in slightly larger fractionation of −5.93 ± 0.61 (Figure 2B). At higher rates where electron donor and acceptor and not initially limiting, like those here, fractionation approaches a minimum near 4 (Harrison and Thode, 1958), perhaps reflecting the fractionation associated with converting sulfate to sulfite. As rates slow, fractionation can range up to >66 (Sim et al., 2011a,b; Leavitt et al., 2013), approaching the predicted equilibrium fractionation (Tudge and Thode, 1950; Farquhar et al., 2003; Johnston et al., 2007), presumably by a relaxation of the thermodynamic driving force (Thullner et al., 2008) For the purposes of this study, we take these fractionations

The isotopic consequences of sulfite and thiosulfate reduction

as a representative baseline for the growth of G20 under sulfate replete conditions. These data are then directly comparable to growth on the other terminal electron acceptors tested here under similar closed system conditions with initially non-limiting electron donor and acceptor. These experiments also serve to extend the catalog of minor sulfur isotopic data for sulfate reduction. From earlier works, it is clear that the MSR process is broadly characterized as having highly variable 34 ε effects with 33 λ that is exclusively less than equilibrium predictions of 0.515. Minor isotope fractionations range down to less than 0.510 with a mean near 0.512 (Farquhar et al., 2003; Johnston et al., 2005, 2007; Canfield et al., 2010; Bradley et al., 2011; Johnston, 2011; Sim et al., 2011a,b, 2012; Leavitt et al., 2013) showing significant co-variance with csSRR (Sim et al., 2011b; Leavitt et al., 2013) Consistent with these findings, the sulfate reduction experiments from this work yield calculated 33 λ of 0.508 ± 0.002 and 0.511 ± 0.001 for lactate and formate, respectively (Table 1). It is important to note that the error on 33 λ is heavily (and non-linearly) dependent on 34 ε, meaning that at low 34 ε, the error is larger and the 33 λ less uniquely resolvable. Here we use updated error propagation equations derived previously (Johnston et al., 2007). Along with these new data, we recalculated (i.e., normalized) and compiled published pure culture results from both open- and closed-system sulfate reduction experiments (Figure 3B). The compilation highlights a number of key observations. Foremost among these is the resolvable triple isotope trend where elevated 33 λ corresponds to larger 34 ε fractionation, as recently highlighted in both open and closed system studies (Sim et al., 2011b; Leavitt et al., 2013). Despite this trajectory toward low temperature thermodynamic equilibrium, such theoretical values have not yet been observed in a microbial experiment (Canfield et al., 2010; Sim et al., 2011a,b; Ono et al., 2012; Leavitt et al., 2013). In light of this clear triple isotope relationship, it is important to note that this compilation represents a variety of different MSR strains over a range of conditions. For deeper physiological understanding, further continuous culture (open-system) and theoretical work are necessary. Still, a great deal of isotopic behavior is shared among the different experimental approaches and is likely universal to MSRs.

THIOSULFATE REDUCTION

FIGURE 2 | Data from sulfate reduction experiments with lactate (red) or formate (blue). (A) The loss of sulfate is accounted for with the ingrowth of sulfide. Data reflect various time points throughout the experiment and the line corresponds to perfect closure of elemental mass balance (theory). (B) The calculated fractionation factor (34 ε) relating sulfate and sulfide using f and the equations from the text. The isotopic residuals (to satisfy the calculated mass-imbalance) on the sulfate experiments are 0.18 and 0.42 in δ34 S and 0.007 and 0.017 in 33 S for formate and lactate, respectively.

Frontiers in Microbiology | Microbiological Chemistry

The thiosulfate reduction experiment with G20 carried straightforward geochemical results, with thiosulfate reduced to hydrogen sulfide (Figure 4A). This is consistent with previous reports for three other strains of sulfate reducers (D. desulfuricans, D. sulfoexigens, and D. multivorans) grown on thiosulfate (Habicht et al., 1998; Smock et al., 1998). Over the course of our experiment, 15% of the thiosulfate sulfur was reduced to sulfide, leading to cell specific thiosulfate reduction rates of up to 265 f mol per cell per day during exponential phase (Table 2). However, predictions from earlier work on MSR suggest that sulfite serves as an intermediate between thiosulfate and sulfide (essentially the liberation and subsequent reduction of the sulfonate S) (Smock et al., 1998). Although methods for quantifying sulfite were employed in these experiments, sulfite was never above the detection limit (

Multiple sulfur isotope signatures of sulfite and thiosulfate reduction by the model dissimilatory sulfate-reducer, Desulfovibrio alaskensis str. G20.

Dissimilatory sulfate reduction serves as a key metabolic carbon remineralization process in anoxic marine environments. Sulfate reducing microorganis...
2MB Sizes 1 Downloads 6 Views